Dsb Formation in the SHuffle Strains
SHuffle strains contain a unique trio of modifications that produce robust disulfide bond formation in the cytoplasm.
First, the cytoplasmic environment is altered by elimination of glutathione reductase (gor gene product) and thioredoxin reductase (trxB gene product). Since the combination of gor and trxB deletions are lethal, a suppressor mutation in the ahpC gene is necessary for the SHuffle (and Origami) strains to maintain viability.
Second, SHuffle strains are uniquely engineered to overexpress DsbC within the cytoplasm. The DsbC enzyme acts as a disulfide bond isomerase and “shuffles” mis-oxidized cysteine pairs allowing the recombinant target protein to achieve its properly folded confirmation. Due to the action of DsbC in the SHuffle cell, less target protein proceeds down the paths of protease degradation or inclusion body formation.
Finally, the SHuffle strains were engineered from robust parent strains capable of tightly controlled protein expression. For example, SHuffle T7 Express (B strain) and SHuffle T7 (K-12 strain) both express the T7 RNA polymerase from the lac operon, whereas most other T7 expression strains utilize the DE3 prophage.
T7 expression in DE3 strains is known to be somewhat uncontrolled and this can have very detrimental effects on cell growth and protein yield when the target protein is even mildly toxic, which is a common occurrence with heterologous dsb-containing proteins. If strictly controlled T7 expression is required, then lysY strains are also available.
The lysY gene product is a variant of T7 lysozyme containing a mutation that eliminates “lysozyme” function on the E. coli cell wall, but the ability of LysY to inhibit T7 RNA polymerase function is unaffected. During the pre-induction phase, a constant low level of LysY inhibitor protein is produced to inactivate any basal expression of T7 RNA polymerase.